argonaute 2 complexes selectively protect the circulating micrornas in cell-secreted microvesicles微核醣核酸在细胞分泌argonaute 2复合物有选择地保护循环微泡.pdf

Argonaute 2 Complexes Selectively Protect the Circulating MicroRNAs in Cell-Secreted Microvesicles 1 1 1 1 1,2 1 2 1 Limin Li , Dihan Zhu , Lei Huang , Jing Zhang , Zhen Bian , Xi Chen , Yuan Liu , Chen-Yu Zhang *, Ke Zen1* 1Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China, 2 CMBP, Department of Biology, Georgia State University, Atlanta, Georgia, United States of America Abstract Cell-secreted miRNAs are highly stable and can serve as biomarkers for various diseases and signaling molecules in intercellular communication. The mechanism underlying the stability of circulating miRNAs, however, remains incompletely understood. Here we show that Argonaute 2 (Ago2) complexes and microvesicles (MVs) provide specific and non-specific protection for miRNA in cell-secreted MVs, respectively. First, the resistance of MV-encapsulated miRNAs to RNaseA was both depended on intact vesicular structure of MVs and protease-sensitive. Second, an immunoprecipitation assay using a probe complementary to human miR-16, a miRNA primarily located in the MVs and showed a strong, protease-sensitive resistance to RNaseA, identified Ago2 as a major miR-16-associated protein. Compared with protein-free miR-16, Ago2- associated miR-16 was resistant to RNaseA in a dose- and time-dependent fashion. Third, when the miR-16/Ago2 complex was disrupted by trypaflavine, the resistance of miR-16 to RNaseA was decreased. In contrast, when the association of miR- 16 with the Ago2 complexes was increased during cell apoptosis, although the total amount of miR-16 and Ago2 remained